Illumina HiSeq 4000 paired end sequencing; RNA-seq of wheat lodicules in four varieties at pre-flowering and early flowering stages
RNA-seq of wheat lodicules in four varieties at pre-flowering and early flowering stages
The plant material consisted of two highly-chasmogamous (HCH) cultivars: Piko (DE, 2009, CWW-3319.5/3/Kraka//Maris Huntsman/Fruhgold) and Poezja (LT, 2017, STH43/STH248-93-14-326), and two low-chasmogamy (LCH) cultivars: Euforia (PL, 2018, Muszelka//N5113/STH3907) and KWS Dacanto (PL, 2011, Opus/Certo) cultivars of hexaploid winter wheat.In order to identify candidate genes controlling lodicules opening, lodicules of HCH and LCH genotypes were harvested from spikes at pre-flowering (GS95 - ear completely emerged above flag leaf ligule) corresponding to green anther stage (GAS) and early flowering (GS61 - start of flowering (first anthers visible) referred as yellow anther stage (YAS) (5, 32). The samples were collected in three biological replicates. Approximately 50-100 lodicules were samples from five to seven individual spikes for each of the three replicates. The materials were immediately submerged in RNAlater™ Stabilization Solution (Ambion, Thermo Fisher Scientific, Waltham, MA, USA) and kept at -80˚C until required.  After 6-week vernalization at 4˚C, plants were transferred to a growth chamber (ForClean, ZalMed Sp. z.o.o, Warsaw, Poland) under the following conditions: temperature - 18˚C/15˚C day/night, 14/10 h photoperiod and constant relative humidity of 50%. Plants were cultivated in pots filled with a mixture of peat and perlite (1:1, v/v), three plants per single pot (19 cm diameter). The plants were hand irrigated daily to maintain soil water content close to field capacity. GeneMATRIX Universal RNA Purification kit (EURx, Gdańsk, Poland) was used to isolate total RNA from the preserved lodicules. RNA concentration was measured by means of a Qubit® 2.0 Fluorometer (Invitrogen, Waltham, MA, USA) and its quality and integrity were verified on BioAnalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA) using Agilent RNA 6000 Nano Kit.  Twenty four strand-specific cDNA libraries (for four cultivars, two anthers developmental stages, and three biological replications) were constructed with NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® (New England Biolabs, Ipswich, MA, USA). The Illumina HiSeq 4000 platform in the PE150 mode was used for RNA-Seq and the data were analyzed by Genomed S.A (Warsaw, Poland).